Abstract

   Camel milk exosomes from colostrum (C) and mature milk (M) were
   characterized using proteomics and lipidomics to elucidate their
   compositional and functional differences. Isolated exosomes exhibited
   round and cup-like concave spheres of 30 to 150 nm and higher TSG101
   expression in colostrum. Proteomic analysis identified 2479 proteins,
   with 843 differentially expressed (357 upregulated, 486 downregulated)
   between C and M, enriched in PI3K-AKT signaling, endocytosis, and
   complement/coagulation pathways, suggesting roles in immune regulation
   and neonatal development. Lipidomics revealed 1783 lipid species (37
   subclasses), with 149 significantly altered between C and M, including
   SM (d42:4; FC = 221.887) and LPE (18:3; FC = 0.139). Colostrum exosomes
   were enriched in sphingomyelins and glycerophospholipids, while mature
   milk contained higher lysophospholipids, reflecting stage-specific
   nutritional adaptations. These findings provide novel insights into
   camel milk exosome biology, highlighting their potential as biomarkers
   and functional food components for infant nutrition and health
   applications.

   Keywords: Camel milk, Exosome, Proteomics, Lipidomics

Highlights

     * •
       Camel colostrum and mature milk exosomes were round and cup-like
       concave spheres of 30 to 150 nm.
     * •
       There were 843 and 149 differentially exosome proteins and lipid
       species between camel colostrum and mature milk.
     * •
       Critical lipid subclasses were triglycerides, phosphatidylcholines,
       and phosphatidyl ethanolamines.
     * •
       Camel milk exosomes could decrease insulin resistance through the
       PI3K-AKT signaling.

1. Introduction

   Milk is a biological fluid rich in nutrients needed for the growth and
   development of young animals, containing protein, fat, minerals,
   vitamins, and various active ingredients. Due to its relatively
   complete nutritional structure, milk is the most complete food for
   digestion and absorption by newborns. The nutritional composition in
   milk play essential roles in growth, development, gut microbiota
   regulation, and disease prevention ([29]Chen et al., 2021).

   Exosomes are a type of membranous microvesicle widely present in
   various bodily fluids, including milk. Milk contains a high abundance
   of exosomes, which are produced by mammary glands, with a particle size
   ranging from 50 to 150 nm ([30]Zhao et al., 2021). In 2007,
   milk-derived exosomes were first identified in human breast milk, and
   it was demonstrated that these exosomes could modulate immune responses
   ([31]Admyre et al., 2007). Since then, the interest in milk exosomes
   has expanded to different mammals, including cows ([32]Yang et al.,
   2017), buffalo, goats ([33]Ahmed et al., 2022), camels ([34]Shaban et
   al., 2023), and pigs ([35]Ferreira et al., 2021). Milk exosomes exhibit
   complex compositions, containing not only proteins and lipids but also
   DNA and various RNA species, which may serve as potential biomarkers
   for certain diseases ([36]Valadi et al., 2007). It has been confirmed
   that human milk exosomes are RNA-rich and can be absorbed by infants
   during breastfeeding ([37]Lässer et al., 2011). Bovine milk exosomes
   possess a large content of miRNAs that remain stable in acidic gastric
   conditions, enabling intestinal absorption ([38]Izumi et al., 2015).
   [39]Reinhardt et al. (2012) determined that bovine milk exosomes
   included a large amount of protein, and [40]Samuel et al. (2017)
   further suggested that immune-related proteins from bovine milk
   exosomes could mediate immune responses based on proteomic functional
   enrichment analysis. Yak milk-derived exosomes have been shown to
   enhance cellular tolerance to hypoxic conditions ([41]Gao et al.,
   2019). Similarly, camel milk exosomes exhibit inhibitory effects on
   breast cancer cell proliferation ([42]Badawy et al., 2018).
   Furthermore, porcine milk exosomes contain immune-related miRNAs that
   exert anti-inflammatory effects on small intestinal epithelial cells by
   regulating the NF-κB inflammatory signaling pathway ([43]Xie et al.,
   2019).

   Camel milk, comprising milk from both dromedary camels (Camelus
   dromedarius) and Bactrian camels (Camelus bactrianus), exhibits
   distinct nutritional profiles between the two species. Comparative
   analyses reveal significant interspecies variations in milk
   composition, attributable to a combination of genetic factors
   (breed-specific characteristics), environmental adaptations
   (habitat-specific influences), and management practices (rearing
   conditions) ([44]Alhaj et al., 2022). Notably, camel milk possesses
   exceptional nutritional value and multiple benefits, establishing it as
   a traditional and healthy dietary staple for ethnic minority
   populations in northern China. Unlike other types of animal milk, camel
   milk offers unique advantages including high fat and protein content,
   combined with low cholesterol, low sugar, and abundant polyunsaturated
   fatty acids ([45]Shaban et al., 2023). Research has demonstrated that
   camel milk lacks β-lactoglobulin and contains either no or only trace
   amounts of κ-casein, making it more suitable for individuals with
   bovine milk allergies ([46]Cardoso et al., 2010). The fatty acid
   composition in camel milk is easy to digest and absorb and contains
   more long-chain fatty acids (C14-C18), making it healthier to drink.
   Furthermore, in contrast to cow milk, camel milk is rich in vitamin C,
   serving as the primary source of vitamin C for local herders and
   providing an effective alternative to fruits and vegetables for meeting
   vitamin C requirements ([47]Benmeziane-Derradji, 2021). Moreover,
   research has shown that camel milk contains bioactive components
   including lactoferrin, immunoglobulin, lysozyme, and heavy chain
   antibodies (IgG2 and IgG3), which exhibit multiple therapeutic
   properties such as anti-inflammatory, immunomodulatory, and medicinal
   effects, particularly in alleviating diabetes, liver injury, colitis,
   and kidney disease ([48]Ayoub et al., 2018; [49]He et al., 2022;
   [50]Ming et al., 2020).

   Milk-derived exosomes harbor functional proteins and lipids, as
   evidenced by previous studies human and bovine milk exosomes ([51]Yang
   et al., 2017). Nevertheless, there is limited research on exosome
   proteins and lipids in camel milk. This study aims to systematically
   analyze exosome proteins and lipids in camel colostrum and mature milk
   using proteome and lipidomics methods. The research results will
   enhance our comprehension of proteins and lipids of exosomes from
   camels across different lactation stages and provide a data reference
   for the development of camel milk products, particularly non-allergenic
   functional foods.

2. Materials and methods

2.1. Bactrian camel milk sample collection

   Bactrian camel colostrum (0–7 days postpartum) samples were acquired
   from 24 healthy Bactrian camels of 4–5 years old. Parturition dates
   were recorded, and mature milk samples (120–125 days postpartum) were
   obtained from the same camels. All camels were maintained under natural
   grazing conditions. The Animal Administration and Ethics Committee of
   the Camel Protection Association of Inner Mongolia reviewed and
   approved the experimental protocol (Permit No. 2023-006). To minimize
   individual variation, milk samples from three camels were mixed, and in
   each group (colostrum and mature milk), eight samples were stored for
   subsequent experiments. This study was approved by the Inner Mongolia
   Agricultural University.

2.2. Exosome isolation from milk samples

   Exosomes from camel colostrum and mature milk were extracted based on
   ultracentrifugation method as described by Gao et al. ([52]Gao et al.,
   2019) with simple modification. Briefly, milk samples were first
   centrifuged at 2000g for 30 min at 4 °C to separate milk fat. The
   resulting supernatant was centrifuged at 10,000g for 45 min at 4 °C,
   followed by filtration through a 0.45 μm membrane. Subsequently, the
   filtrate centrifuged at 100,000g for 70 min at 4 °C, and crude milk
   exosomes were re-suspended in 1 × phosphate buffer saline (PBS,
   pH = 7.3) and subjected to a second ultracentrifugation (100,000g for
   70 min). Finally, the purified exosomes were resuspended in 100 μL of
   pre-cooled 1× PBS for downstream analyses, including transmission
   electron microscopy (TEM), particle size measurement, as well as
   protein and lipid extraction.

2.3. Identification and analysis of exosomes

2.3.1. TEM

   Exosome samples were applied to a 400 mesh copper grid for 1 min, and
   the excess floating liquid was absorbed using filter paper. Samples
   were negatively stained with 10 μL of a 2 % uranyl acetate solution
   (w/v; Electron Microscopy) for 30 min, air dried, and viewed using a
   transmission electron microscope (Hitachi H-7500, Hitachi, Japan) at
   100 kv.

2.3.2. NTA

   Based on NanoSight NS300 (NAT 3.1, Malvern Panalytical, UK) system to
   detect the concentration and size distribution of particles according
   to the research ([53]Yang et al., 2017). A volume of 10 μL exosomes was
   diluted to 1:25–1:1000-fold in PBS to maintain a particle count between
   50 and 200 per frame for real-time direct imaging and observation of
   specific exosome within a diameter range of 50–1000 nm.

2.3.3. Western blot (WB)

   Exosome proteins were mixed with the protein loading buffer, and loaded
   on 15 % SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel
   Electrophoresis). Proteins were transferred to a polyvinylidene
   difluoride membrane at 300 mA for 2 h (Bio-Rad Laboratories, Munich,
   Germany). The membranes were soaked in 5 % skim milk TBST (Tris-HCl,
   NaCl, Tween 20) with the protein side facing upwards and blocked for
   1 h. Blots were incubated with tumor-sensitive gene 101 protein TSG101
   ([54]ABP56454, Abbkine, USA; dilution ratio was 1:1000) overnight at
   4 °C and washed three times with TBST at room temperature. Then, the
   blots were incubated with goat anti-rabbit secondary antibody (A9169,
   Sigma, USA; dilution ratio was 1:5000) at room temperature for 1 h,
   followed by three washes. After coloring with Tianneng Western blot
   (WB) ultra-sensitive chemiluminescence solution (180–501, Tanon,
   Shanghai), imaging was performed using a VersaDoc MP4000 imaging system
   (Bio-Rad, Hercules, CA, USA).

2.4. Proteomic analysis of camel milk exosomes

2.4.1. Protein extraction and digestion

   Camel milk-derived exosomes were lysed using SDT buffer (4 % SDS,
   100 mM DTT, 150 mM Tris-HCl pH 8.0) and were further sonicated. The
   lysates were centrifuged at 14,000g for 40 min, and the protein
   concentration in supernatant was determined (using the BCA Protein
   Assay Kit, Bio-Rad, USA). A mass of 20 μg protein from each sample was
   mixed with an appropriate amount of 5× loading buffer solution and then
   subjected to SDS-PAGE (4 %–20 % preformed gradient gel, 180 V, 45 min).
   Coomassie Brilliant Blue R-250 staining was performed.

   Protein digestion was completed based on the method of filter-aided
   proteome preparation ([55]Wisniewski et al., 2009), and were
   fractionated using a Thermo Scientific™ Pierce™ High pH Reversed-Phase
   Peptide Fractionation Kit. The C18 Cartridges (Empore™ SPE, Sigma) was
   used to desalt and reconstituted in 40 μL of 0.1 % (v/v) formic acid.

2.4.2. Liquid chromatography−MS/MS analysis

   LC-MS/MS analysis was performed using a Q-Exactive HF-X mass
   spectrometer (Thermo Scientific) with data-independent acquisition
   (DIA) mode. Detection mode: positive ion, primary mass spectrometry
   scanning range: 350–1800 m/z, mass spectrometry resolution: 120000 (@
   m/z 200), AGC target: 3e6, Maximum IT: 30 ms. MS2 used to DIA data
   acquisition mode, with 44 DIA acquisition windows set, mass
   spectrometry resolution: 30000 (@ m/z 200), AGC target: 3e6, Maximum
   IT: auto, MS2 Activation Type: HCD, Normalized conflict energy: 30 eV.
   DIA data were analyzed with Spectronaut TM 14.4.200727.47784. The
   software parameters were set as follows: the retention time prediction
   type was set to dynamic iRT, interference on MS2 level correction was
   enabled, and cross-run normalization was enabled. All results were
   filtered using a Q-value cutoff of 0.01 (corresponding to FDR < 1 %).

2.5. Lipidomic analysis of camel milk exosome

2.5.1. Lipid extraction

   The lipids of camel milk-derived exosomes were isolated with methyl
   tert-butyl ether (MTBE) according to the procedure of [56]Wang et al.
   (2022), with minor variations. Milk samples were mixed with an internal
   lipid standard mixture and MTBE (methanol (v/v, 5:1). After vortex
   mixing for 60 s, the samples were sonicated in a low-temperature water
   bath for 20 min and centrifuged at 10 °C for 15 min at 14,000g, and
   then collected the upper organic phase. The lipid extraction was
   redissolved using 200 μL 90 % isopropanol/acetonitrile solution and
   centrifuged at 10 °C for 15 min at 14,000g before mass spectrometry
   analysis.

2.5.2. UHPLC-Q-Exactive conditions

   Chromatographic analysis was using Ultra High-Performance Liquid
   Chromatography System (UHPLC Nexera LC-30A) with a Kinetex C18 column.
   Mobile phase A consisted of acetonitrile aqueous solution
   (acetonitrile:water = 6:4, v/v). Mobile phase B consisted of an
   acetonitrile-isopropanol solution (acetonitrile:isopropanol = 1:9,
   v/v). Injection volume wa 2 μL, column temperature was 45 °C and low
   rate was 300 μL/min. The elution process was as follows: 0–2 min, 30 %
   B; 2–25 min, 100 % B; 25–35 min. 30 % B. The injection volume was 2 μL
   in positive or negative mode.

   The analyses were performed using electrospray ionization (ESI) in both
   positive and negative ion modes. After UHPLC separation, the samples
   were subjected to mass spectrometry analysis using a Q Exactive series
   mass spectrometer (Thermo Scientific™). The ESI source conditions were
   set as follows: Heater Temp 300 °C, Sheath Gas Flow rate 45 arb, Aux
   Gas Flow Rate15 arb, Sweep Gas Flow Rate 1arb, spray voltage 3.0KV,
   Capillary Temp 350 °C, S-Lens RF Level 50 %, MS1scan ranges: 200–1800.
   The mass-to-charge ratios (m/z) of lipid molecules and their fragments
   were acquired using the following parameters: Ten fragment spectra (MS2
   scan, HCD) were collected following each full scan. MS1 has a
   resolution of 70,000 at M/Z 200, MS2 has a resolution of 17,500 at M/Z
   200.

2.6. Statistics and Bioinformatic analysis

   The significance of differences in exosome size and concentration was
   evaluated using one-way parametric analysis followed by Tukey's post
   hoc test using GraphPad Prism software (Inc., La Jolla, CA) with
   P < 0.05 considered as statistically significant. The graphical
   interface was illustrated using R software. The differentially
   expressed exosome proteins were screened via the Student's t-test and
   fold change (FC) values (upregulation greater than 1.5 times or
   downregulation less than 0.67 times). The screening criteria of
   significantly differentially expressed lipids were set to P < 0.05,
   variable importance in projection (VIP) > 1, and either FC > 1.5
   (upregulated) or FC < 0.67 (downregulated). The statistics function
   ‘prcomp’ within R ([57]https://www.r-project.org) ([58]Chen et al.,
   2021) was used to complete orthogonal partial least squares
   discriminant analysis (OPLS-DA). The identified proteins between camel
   colostrum and mature milk exosomes were displayed in the Venn plot.
   Hierarchical clusters and volcano plots were prepared using the R
   package ‘ComplexHeatmap’ (v3.5.0.) and ‘ggplot2′ (v3.5.0). The
   functional proteins from camel colostrum and mature milk exosomes were
   analyzed using gene ontology (GO) annotation
   ([59]https://geneontology.org/) and the Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathway ([60]https://www.genome.jp/kegg/), respectively.
   Furtherly, the protein-protein interaction (PPI) network was analyzed
   using STRING ([61]https://string-db.org/) and visualized the results in
   Cytocscape V3.10.3 ([62]Shannon et al., 2003). The top 10 hub proteins
   were identified via CytoHubba. Additionally, the lipid-related pathways
   were characterized through lipid pathway enrichment analysis (LIPEA)
   ([63]https://hyperlipea.org/home) to elucidate their biological
   relevance.

3. Results and discussion

3.1. Microstructure and WB analysis of camel colostrum and mature milk
exosomes

   Camel colostrum and mature milk exosomes were characterized using WB
   ([64]Fig. 1A), TEM ([65]Fig. 1B–C), and nanoparticle tracking analysis
   (NTA) ([66]Fig. 1D–F). CD63, TSG101, CD8, CD81, Alix, and flotillin 1
   were widely used as index proteins in WB ([67]Lötvall et al., 2014). In
   this study, the exosomal marker TSG101 was confirmed in both colostrum
   and mature milk, and its expression was higher in the C group
   (colostrum) compared with the M group (mature milk) ([68]Fig. 1A). The
   TEM examination revealed that the exosomes had typical round and
   cup-like concave spheres ([69]Fig. 1B–C). The NTA analysis clearly
   showed that the exosomes extracted from camel colostrum and mature milk
   varied between 30 and 150 nm ([70]Fig. 1F). The camel colostrum
   exosomes (73.75 ± 1.76 nm) were smaller than mature milk exosomes
   (75.12 ± 0.59 nm) (P > 0.05). This result was up to twice the size of
   previously reported mid-lactation milk exosomes in dromedary camels
   (30 nm) ([71]Yassin et al., 2016), two times smaller than those in
   porcine milk (average size 152 nm) ([72]Ferreira et al., 2021),
   slightly larger than human milk (50 nm) ([73]Admyre et al., 2007). The
   concentration of colostrum exosomes was higher (5.02 × 10^12
   particle/mL) than mature milk (3.62 × 10^12 particle/mL) (P > 0.05).
   [74]Shijie (2020) reported that the concentration of exosomes in
   Bactrian camels was about 1.7 × 10^12 particle/mL, which was
   significantly lower than our result. These differences might be because
   of the camel species, age, parity, environment, feeding, and lactation
   period.

Fig. 1.

   [75]Fig. 1
   [76]Open in a new tab

   Identification of exosomal marker TSG101 in the camel colostrum (C),
   mature milk (M), supernatant exosomes (SE), 293T control by WB (A). TEM
   scanning of camel colostrum (B) and mature milk (C) exosomes. The scale
   bar corresponds to 100 nm. NTA analysis for size distribution (D,F) and
   concentration (E) in camel colostrum and mature milk.

3.2. Characterization of exosome proteins in camel colostrum and mature milk

   There 2479 kinds of milk-derived exosome proteins were identified in
   camel colostrum and mature milk, including 2398 shared proteins, 220
   colostrum-specific proteins, and 131 mature milk-specific proteins
   ([77]Fig. 2A). Exosome marker proteins including transmembrane proteins
   (CD9, CD63, and CD81), heat shock proteins (HSC70 and HSC90), membrane
   transporters (GTPases), and lipid binding proteins were detected in
   both camel colostrum and mature milk exosomes. Moreover, typical milk
   proteins, including butyrophilin and lactadherin, were present in camel
   milk exosomes. Butyrophilin stimulates the secretion of lipid droplets
   in milk, and its extracellular domain may have receptor functions.
   Lactadherin is a membrane glycoprotein that promotes phagocytosis of
   apoptotic cells. These proteins were first reported in human and bovine
   milk exosomes by [78]Admyre et al. (2007) and [79]Reinhardt et al.
   (2012). We also found Rab proteins in camel milk exosomes, which play
   an important role in vesicle fusion and trafficking events. Previous
   research has shown that Rab proteins can regulate cellular growth and
   development processes ([80]Amir et al., 2016). These specific exosome
   proteins in camel milk exosomes could be used as potential biomarkers
   for the development of functional foods. Through our research, a large
   number of camel milk exosome proteins were identified, which will
   provide data to support further elucidating the composition, potential
   biological functions, and utilization of camel milk exosomes.

Fig. 2.

   [81]Fig. 2
   [82]Open in a new tab

   Identified proteins in camel colostrum and mature milk exosomes. Venn
   diagram of the exosome proteins in camel colostrum and mature milk (A).
   Bar chart of differentially expressed exosome proteins (B). Clustering
   heatmap of differentially expressed exosome proteins between camel
   colostrum and mature milk (C). C = camel colostrum milk; M = camel
   mature milk.

   Furthermore, the differentially expressed exosome proteins in camel
   colostrum and mature milk were evaluated ([83]Fig. 2B–C). A total of
   843 differentially expressed milk exosome proteins were found
   (P < 0.05) (357 upregulated and 486 downregulated) in camel colostrum
   and mature milk based on the criteria of FC >1.5 (Table S1). The
   volcano plots of log[2](FC) (x-axis) and their associated
   −log[10](P-values) (y-axis) of all identified proteins in camel
   colostrum and mature milk were provided in Fig. S1. In our research,
   differences between the exosome proteomes in camel colostrum and mature
   milk were still greater than those found comparing the proteome of
   bovine mature milk and colostrum ([84]Yang et al., 2017), human mature
   milk and colostrum ([85]Yang et al., 2017), and porcine mature milk and
   colostrum ([86]Ferreira et al., 2021). Additionally, some proteins
   exhibited more than 10 times and less than 0.1 times, respectively, in
   comparison with camel colostrum and mature milk exosomes ([87]Table 1).
   Apoptosis, oxidative stress, inflammatory response, and immune-related
   proteins, such as protein glutathione peroxidase 3, heat shock 70 kDa
   protein 13, clusterin, IgG receptor FcRn large subunit p51 isoform X2,
   leucine-rich repeat-containing protein 10B were highly expressed in
   colostrum compared with mature milk exosomes. These proteins are
   significant in the healthy growth of neonates. This result is similar
   to previous reports in which exosome proteins derived from human,
   bovine, and porcine colostrum were significantly enriched in the immune
   response ([88]Admyre et al., 2007; [89]Ferreira et al., 2021;
   [90]Shaban et al., 2023).

Table 1.

   Differentially expressed exosome proteins between camel colostrum (C)
   and mature milk (M).
   Uniprot Accession Protein name FC value(C/M) p-value Regulated type
   A0A8B8RDZ4 methionine aminopeptidase 1 55.943 3.040E-02 up
   A0A8B6YPU0 colostrum trypsin inhibitor 29.869 3.316E-02 up
   A0A8B8U7E8 epidermal growth factor receptor substrate 15 isoform X1
   29.072 1.744E-03 up
   A0A8B8S9C1 clusterin 27.351 5.962E-03 up
   A0A8B6Y776 HLA class II histocompatibility antigen gamma chain isoform
   X1 19.780 2.676E-04 up
   A0A8B6YEM0 heat shock 70 kDa protein 13 17.078 1.803E-05 up
   A0A8B6YAG0 glutathione peroxidase 3 15.498 1.138E-02 up
   A0A8B8TBK9 tetraspanin-12 14.602 4.499E-02 up
   A0A8B8SK91 biglycan 14.349 4.765E-02 up
   A0A8B6YFL2 stromal cell-derived factor 2 isoform X2 14.168 2.249E-02
   A0A8B7KBK0 IgG receptor FcRn large subunit p51 isoform X2 12.193
   9.301E-05 up
   A0A8B6YQ24 leucine-rich repeat-containing protein 10B 11.208 9.263E-03
   up
   A0A8B8TKG6 procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 isoform X1
   10.640 4.956E-02 up
   A0A8B6YP51 quinone oxidoreductase PIG3 0.098 2.791E-04 down
   A0A8B6YPH5 protein O-glucosyltransferase 2 isoform X2 0.095 5.615E-05
   down
   A0A8B8RY85 major facilitator superfamily domain-containing protein 4A
   isoform X1 0.088 6.286E-07 down
   [91]S9XED8 small integral membrane protein 1 0.078 5.655E-05 down
   A0A8B8UIC1 LOW QUALITY PROTEIN: CMT1A duplicated region transcript 1
   protein 0.077 4.124E-02 down
   A0A8B8SE62 amyloid-like protein 2 isoform X1 0.060 3.789E-05 down
   A0A8B8TYV5 aspartate aminotransferase, cytoplasmic 0.053 3.557E-02 down
   A0A8B6YRE3 laminin subunit gamma-1 0.047 7.159E-03 down
   A0A8B8TRK7 polypeptide N-acetylgalactosaminyltransferase 18 0.037
   1.294E-04 down
   [92]Open in a new tab

3.3. GO analysis of the differentially expressed exosome proteins

   Research on exosome proteomics in both colostrum and mature milk from
   bovine and humans indicated that exosome proteins are mainly involved
   in ribosomes and regulating the actin cytoskeleton, proteins regulating
   cell growth, and so on ([93]Samuel et al., 2017; [94]Yang et al.,
   2017). Gene Ontology (GO) analysis was conducted to assess functional
   differences, with the top 20 significantly enriched terms presented in
   [95]Fig. 3. The most differential GO terms among biological processes
   were regulation of multicellular organismal process (GO.0051241) and
   regulation of vesicle−mediated transport (GO.0060627). The molecular
   functions of these proteins were predominantly concentrated in myosin
   binding (GO.0017022) and GDP binding (GO.0019003). Regarding cellular
   components, exosome proteins mainly originated from the extracellular
   region (GO.0005576), extracellular space (GO.0005615), and
   extracellular exosome (GO.0070062), and these functional categories may
   be related to forming vesicular structures in the exosomes.

Fig. 3.

   [96]Fig. 3
   [97]Open in a new tab

   GO annotation of differentially expressed exosome proteins in camel
   colostrum and mature milk. BP, Biological processes; MF, Molecular
   functions; CC, Cellular components.

3.4. KEGG pathway analysis of the differentially expressed exosome proteins

   The top 20 KEGG pathways and pathway enrichment of differentially
   expressed proteins are illustrated in [98]Fig. 4A and B. Most
   differentially expressed milk exosome proteins were involved in
   endocytosis, complement, and coagulation cascades, regulation of the
   actin cytoskeleton, PI3K-Akt signaling pathway, and ribosomes, while
   some milk exosome proteins were associated with leukocyte
   transendothelial migration, ras signaling pathway, rap1 signaling
   pathway, tight junction, MAPK signaling pathway, and platelet
   activation. Previous studies have also confirmed that most exosome
   proteins from human and bovine milk are involved in these KEGG pathways
   ([99]Yassin et al., 2016; [100]Zhao et al., 2021). The endocytic
   pathway is “a bridge” for exchanging information between cells and the
   external environment and is responsible for delivering membrane
   proteins and phospholipids ([101]Doherty & Mcmahon, 2009). These
   differentially expressed milk exosome proteins can ensure the normal
   progress of various cellular biological processes, such as nutrient
   absorption, cell migration, cell division, and growth factor receptor
   regulation ([102]Scott et al., 2014).

Fig. 4.

   [103]Fig. 4
   [104]Open in a new tab

   Differentially expressed proteins of KEGG enrichment between camel
   colostrum and mature milk exosomes. The top 20 differentially expressed
   proteins of KEGG (A). Pathway enrichment of differentially expressed
   proteins upregulated and downregulated (B).

   There were 33 differentially milk exosome proteins enriched in the
   complement and coagulation cascades (Fig. S3), which were important
   components of the immune system. Previous studies reported that camel
   milk exosomes exhibit antibacterial and antifungal effects, modulated
   oxidative stress as well as may inhibit the proliferation of various
   cancer cell lines, including HepG2, HepaRG, CaCo2, Hl60, and PANC1
   ([105]Badawy et al., 2018; [106]El-kattawy et al., 2021; [107]Ibrahim
   et al., 2019; [108]Shaban et al., 2023). These proteins, which
   participate in complement and coagulation cascades, may play a
   preventive and alleviating role in diseases and inflammation. The
   PI3K-AKT signaling pathway, which plays a pivotal role in insulin
   stimulation and cell survival ([109]Huang et al., 2018), is
   participated in metabolic diseases such as diabetes. Some studies
   demonstrated the favorable effects of camel milk on diabetes through
   anti-inflammatory and antioxidant activities ([110]Mirmiran et al.,
   2017). Our findings further confirmed that camel milk exosome proteins
   might reduce insulin resistance by the PI3K-AKT signaling pathway.

   Furthermore, the endocytosis pathway (Fig. S2) and the complement and
   coagulation cascades pathway exhibited the highest number of
   differentially expressed proteins and the most significant enrichment.
   Endocytosis is a critical component of cancer hallmarks, influencing
   drug stability, therapeutic response, and immune cell function.
   Meanwhile, the complement and coagulation cascades pathway, part of the
   innate immune system, generates diverse effector molecules through
   nonspecific recognition mechanisms, playing a central role in pathogen
   clearance, inflammatory regulation, and maintenance of coagulation
   function ([111]Zhang et al., 2020). The significant enrichment of
   differentially expressed proteins in these two key pathways, as
   identified in this study, may suggest functional differences in immune
   regulation, anti-inflammatory effects, and potential antitumor activity
   between camel colostrum-derived exosomes and mature milk-derived
   exosomes.

   To further explore functional interactions among differentially
   expressed proteins, we constructed a PPI network using STRING. The
   network contained 740 nodes and 4004 edges, with an average node degree
   of 10.8 (Fig. S4). The top 10 hub proteins were evaluated using three
   centrality methods (Degree, EPC, and MCC) in the CytoHubba plugin,
   including Ribosomal protein S27A(RPS27A, albumin (ALB), ubiquitin
   (UBB), clathrin heavy chain 1 (CLTC), ras homolog family member A
   (RHOA), growth factor receptor-bound protein2 (GRB2), apolipoprotein
   B(APOB), catenin beta-1 (CTNNB1), and integrin beta-1 (ITGB1). These
   hubs are functionally linked to endocytosis, complement cascades, and
   PI3K-AKT signaling, consistent with KEGG enrichment results. Notably,
   ALB and ITGB1 were more abundant in camel colostrum-derived exsomes
   than in mature milk exosomes. Both proteins exhibit complementary
   anti-inflammatory and antioxidant properties. ALB plays regulatory
   roles in antioxidant and anti-inflammatory functions ([112]Bihari et
   al., 2020; [113]Tanik et al., 2020). This aligns with the observed
   downregulation of pro-inflammatory genes (TNFα, NFkB, TGFβ1) in HepaRG
   cells treated with colostrum-derived exosomes ([114]El-kattawy et al.,
   2021), suggesting that ALB may synergistically suppress NF-κB signaling
   and oxidative stress in the tumor microenvironment. Moreover, the high
   abundance of lactoferrin in camel colostrum exosomes could potentiate
   ALB's iron-chelating and antioxidant effects, contributing to the
   reported anti-cancer properties of camel milk exosomes ([115]Badawy,
   El-Magd and AlSadrah, 2018, [116]Badawy, Othman and El-Magd, 2021;
   [117]El-kattawy et al., 2021). ITGB1 overexpression has been shown to
   reduce pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and reactive
   oxygen species (ROS) production in LPS-injured cells ([118]Qi et al.,
   2024; [119]Song et al., 2019), suggesting its crucial involvement in
   tissue repair mechanisms. The remaining hub proteins collectively
   contribute to essential cellular processes including protein synthesis
   (RPS27A), lipid metabolism (APOB) ([120]Behbodikhah et al., 2021),
   vesicular trafficking and endocytosis (CLTC), growth factor signal
   transduction (GRB2) ([121]Wang et al., 2024), and Wnt signaling and
   cell adhesion (CTNNB1) ([122]Van der Wal & Van Amerongen, 2020). This
   network analysis not only confirms the KEGG pathway enrichment results
   but also identifies key molecular players that may underlie the
   developmental and protective functions of camel milk exosomes.

3.5. Characterization of exosomes lipids in camel colostrum and mature milk

   The lipidomic analysis of camel colostrum and mature milk exosomes was
   performed in positive (POS) and negative ion (NEG) modes. A total of
   1783 lipid species were identified and classified into 37 different
   lipid subclasses ([123]Fig. 5A and C), including 350 TGs, 149 DGs, 26
   PCs, 28 PEs, 44 PAs, 61 PSs, 28 LPEs, 3 SMs, 44 SPHs, 174 Cers, 42
   CerPs, 77 Hex1Cers, 32 Hex2Cers, and 34 WEs. The qualitative and
   quantitative lipid results are given in Table S2. The top five lipid
   classes included triglycerides (TGs,19.63 %), phosphatidylcholines
   (PCs, 15.87 %), phosphatidyl ethanolamines (PEs, 11.95 %), ceramides
   (Cers, 9.76 %), and diacylglycerols (DGs, 8.36 %) (Fig. S5).

Fig. 5.

   [124]Fig. 5
   [125]Open in a new tab

   Identified lipid subclasses in camel colostrum and mature milk
   exosomes. Number of lipids in each subclass (A). The proportion of
   various lipids in camel colostrum exosomes (B). Comparison of the
   identified lipid numbers between POS and NEG ion modes (C). The
   proportion of various lipids in camel mature milk exosomes (D).

   Distinct lipid composition profiles were observed between camel
   colostrum and mature milk exosomes ([126]Fig. 5B and D). In colostrum
   exosomes, WE, PC SPH, and PE were the predominant lipid components,
   while PC, PE, WE, and PA were the main lipid components in mature milk
   exosomes. These were completely different from the content of
   lipidomics in camel milk, and TG was the highest lipid in mammalian
   milk, such as camel, human, cow, buffalo, horse, goat, and pig ([127]Wu
   et al., 2023). The level of PC in camel mature exosomes (19.71 %) was
   higher than colostrum milk exosomes (14.02 %), and this was higher than
   that of the content in camel milk (11.49 %) ([128]Wu et al., 2023).
   Research reported that PC was the main source of choline ([129]Zou et
   al., 2013), which is crucial for the growth and development of infants.
   PE was reported to be enriched in camel colostrum and mature milk
   exosomes, 11.41 % and 13.73 %, respectively, which is similar to the
   content in camel milk ([130]Wu et al., 2023). Previous studies
   suggested that PE was an essential composition of mammalian cell
   membranes and it has been shown to enhance brain development
   ([131]Zheng et al., 2023). Therefore, the high levels of PE compounds
   in camel milk exosomes, which were together with camel milk, may play
   an important role in the development of the nervous system.
   Interestingly, a high content of PS in camel colostrum (6.98 %) and
   mature milk (8.38 %) exosomes was found, and this may be related to
   enhanced long-term health outcomes since PS can improve nerve cell
   function and facilitate brain memory function ([132]Chizhikov et al.,
   2020). In addition, the level of SPH was significantly higher in camel
   colostrum exosomes (13.36 %) compared to mature milk exosomes,
   suggesting this lipid enrichment may accelerate neonatal immune defense
   development ([133]Nilsson et al., 2021). In contrast, the
   concentrations of TC, PA, and CerP showed no significant differences
   between the colostrum and mature milk exosome groups ([134]Fig. 5B and
   D).

3.6. Differential analysis of lipid species in camel milk exosomes

   A supervised OPLS-DA model was used to visualize the separation of
   lipids in camel colostrum and mature milk exosomes. Based on the
   OPLS-DA score plots ([135]Fig. 6A), lipid extracts in the colostrum and
   mature milk groups displayed a high degree of separation. To better
   screen for potential lipid markers, the criteria set were OPLS-DA VIP
   value>1, P < 0.05, and either FC > 1.5 or FC < 0.67. Differential lipid
   analysis revealed 149 significantly different lipid species belonging
   to 16 lipid subclasses between the colostrum and mature milk groups
   (Table S3). This result suggested that most of the lipids in exosomes
   were the same in camel colostrum and mature milk exosomes. Furthermore,
   a volcano plot was constructed to exhibit the differences in lipids and
   to evaluate their statistical significance ([136]Fig. 6B). This
   included 61 upregulated and 88 downregulated significantly expressed
   lipids in the colostrum compared with the mature milk group, while SM
   (d42:4) and LPE (18:3) showed the highest (221.887) and lowest (0.139)
   fold change values. This indicated that lipids with the highest
   multiple of differences are not the most abundant lipids, but the
   changes in their contents in colostrum and mature milk may have some
   effects on the chemical composition and nutritional value of camel milk
   exosome lipids ([137]Wang et al., 2023). The LIPEA result showed that
   the main pathways involved in differential lipid molecules include
   glycerophospholipid metabolism, sphingolipid signaling pathway, and
   ferroptosis (Fig. S6). These pathways may drive the functional
   divergence between camel colostrum and mature milk exosomes.

Fig. 6.

   [138]Fig. 6
   [139]Open in a new tab

   Analysis of OPLS-DA (A), Volcano plot (B), and heatmap in the camel
   milk exosomes (C).

   C = camel colostrum; M = camel mature milk.

   To better visualize lipid differences between the colostrum and mature
   milk groups, we selected a subset of differentially abundant lipids to
   generate a hierarchical clustering heatmap ([140]Fig. 6C). Camel
   colostrum-enriched lipid profiles revealed a predominance of
   glycerophospholipids (e.g., PE(34:1e)-H, PC(40:0) + HCOO),
   sphingomyelins (SM(d37:1) + HCOO, SM(d44:1) + HCOO), and
   triacylglycerols (TG(52:2) + NH4, TG(54:2) + NH4), suggesting their
   collective roles in enhancing membrane fluidity, supporting neonatal
   neuronal/immune development, and meeting the high energy demands of
   newborns, respectively. These findings underscore the functional
   adaptation of camel colostrum exosomes to early-life nutritional and
   physiological requirements. In contrast, camel mature milk-enriched
   lipids were characterized by elevated levels of lysophospholipids
   (e.g., LPE(18:2)-H, LPC(16:1) + HCOO), reflecting enhanced lipid
   metabolism efficiency, and ether-linked PCs (e.g., PC(18:1e) + H,
   PC(18:2e) + H), which were associated with improved oxidative
   stability. These findings indicate a shift toward lipid species that
   support long-term nutrient absorption and membrane integrity in later
   lactation stages.

4. Conclusions

   This study provides the first comprehensive characterization of
   exosomal proteins and lipids in camel colostrum (C) and mature milk
   (M), revealing significant compositional and functional differences.
   The isolated exosomes exhibited typical morphological features,
   including spherical and cup-shaped structures with diameters ranging
   predominantly between 30 and 150 nm. Comparative analysis revealed 843
   differentially expressed proteins and 1783 lipid species between
   colostrum and mature milk exosomes. Functional investigation of these
   differentially abundant molecules demonstrated their potential roles in
   neonatal development. These findings advance the understanding of camel
   milk exosome biology and offer a foundation for: developing functional
   dairy products by leveraging exosome-derived bioactive components;
   identifying lactation-stage-specific biomarkers for quality control or
   therapeutic applications; exploring interspecies conservation of
   exosome functions in milk. Future studies should validate the
   functional implications of the identified proteins and lipids,
   particularly their roles in infant nutrition and health.

CRediT authorship contribution statement

   Liang Ming: Funding acquisition, Writing – original draft,
   Conceptualization, Formal analysis. Li Yi: Formal analysis, Software,
   Writing – review & editing, Conceptualization. Yisi Ai: Formal
   analysis, Investigation, Methodology. Rimutu Ji: Writing – review &
   editing, Conceptualization, Funding acquisition, Project
   administration.

Declaration of competing interest

   The authors declare that they have no known competing financial
   interests or personal relationships that could have appeared to
   influence the work reported in this paper.

Acknowledgements